Validation of reference genes for the normalization of RT-qPCR gene expression in Bacillus siamensis 1021 grown in different culture media
Abstract
Background and Objectives: House-keeping genes are generally selected as reference genes in gene expression analysis. However, some genes may not be stably expressed across all experimental conditions. Thus, this study aimed to validate seven house-keeping genes for gene expression analysis in Bacillus siamensis 1021.
Materials and Methods: Strain 1021 was grown in potato dextrose broth, nutrient broth and mineral salt medium. Reverse-transcription quantitative PCR was used to determine Cq values of seven reference genes including gyrA, gyrB, ssb and dnaB, rpsU, gat_Yqey and udp in these media. Expression stability of these genes was analyzed, using geNorm and Normfinder applications. The target gene ftsZ was used for assessment of the best candidate genes.
Results: Based on geNorm and Normfinder, ssb was the most-stably expressed gene, while udp was the least-stably expressed gene. Pairwise variation indicated the combination of ssb, gyrA, gyrB and gatB_Yqey was suitable for the normalization of ftsZ expression. ftsZ expression in potato dextrose broth and mineral salt medium was higher than that in nutrient broth. In contrast, the normalization against udp resulted in an under- and overestimation of ftsZ expression in potato dextrose broth and mineral salt medium, respectively.
Conclusion: The combination of ssb, gyrA, gyrB and gatB_Yqey was the best candidate for normalization of target gene expression in B. siamensis 1021 in these media. This study emphasized the significance of reference gene validation for gene expression analysis and provided a guideline for future gene expression studies in B. siamensis.
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30. Higazy NS, Saleh AE, Hassan ZU, Thani RA, Migheli Q, Jaoua S. Investigation and application of Bacillus pumilus QBP344-3 in the control of Aspergillus carbonarius and ochratoxin A contamination. Food Control 2019; 119: 107464.
31. Sun D, Liao J, Sun L, Wang Y, Liu Y, Deng Q, et al. Effect of media and fermentation conditions on surfactin and iturin homologues produced by Bacillus natto NT-6: LC–MS analysis. AMB Express 2019; 9: 120.
32. Xu WF, Ren HS, Ou T, Lei T, Wei JH, Huang CS, et al. Genomic and functional characterization of the endophytic Bacillus subtilis 7PJ-16 strain, a potential biocontrol agent of mulberry fruit sclerotiniose. Microb Ecol 2019; 77: 651-663.
33. Männik J, Walker BE, Männik J. Cell cycle-dependent regulation of FtsZ in Escherichia coli in slow growth conditions. Mol Microbiol 2018; 110: 1030-1044.
34. Holtzendorff J, Partensky F, Jacquet S, Bruyant F, Marie D, Garczarek L, et al. Diel expression of cell cycle-related genes in synchronized cultures of Prochlorococcus sp. strain PCC 9511. J Bacteriol 2001; 183: 915-920.
35. Wu L, Wu H, Chen L, Xie S, Zang H, Borriss R, et al. Bacilysin from Bacillus amyloliquefaciens FZB42 has specific bactericidal activity against harmful algal bloom species. Appl Environ Microbiol 2014; 80: 7512-7520.
2. Suzuki T, Higgins PJ, Crawford DR. Control selection for RNA quantitation. Biotechniques 2000; 29: 332-337.
3. Bai B, Ren J, Bai F, Hao L. Selection and validation of reference genes for gene expression studies in Pseudomonas brassicacearum GS20 using real-time quantitative reverse transcription PCR. PLoS One 2020; 15(1): e0227927.
4. Savli H, Karadenizli A, Kolayli F, Gundes S, Ozbek U, Vahaboglu H. Expression stability of six housekeeping genes: A proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J Med Microbiol 2003; 52: 403-408.
5. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009; 55: 611-622.
6. Köhsler M, Leitsch D, Müller N, Walochnik J. Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp. Sci Rep 2020; 10: 10362.
7. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002; 3: RESEARCH0034.
8. Andersen CL, Jensen JL, Ørntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004; 64: 5245-5250.
9. Sumpavapol P, Tongyonk L, Tanasupawat S, Chokesajjawatee N, Luxananil P, Visessanguan W. Bacillus siamensis sp. nov., isolated from salted crab (poo-khem) in Thailand. Int J Syst Evol Microbiol 2010; 60: 2364-2370.
10. Fan B, Blom J, Klenk HP, Borriss R. Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis form an “Operational Group B. amyloliquefaciens” within the B. subtilis species complex. Front Microbiol 2017; 8: 22.
11. Azabou MC, Gharbi Y, Medhioub I, Ennouri K, Barham H, Tounsi S, et al. The endophytic strain Bacillus velezensis OEE1: An efficient biocontrol agent against Verticillium wilt of olive and a potential plant growth promoting bacteria. Biol Control 2020; 142: 104168.
12. Cui W, He P, Munir S, He P, Li X, Li Y, et al. Efficacy of plant growth promoting bacteria Bacillus amyloliquefaciens B9601-Y2 for biocontrol of southern corn leaf blight. Biol Control 2019; 139: 104080.
13. Liu Y, Teng K, Wang T, Dong E, Zhang M, Tao Y, et al. Antimicrobial Bacillus velezensis HC6: production of three kinds of lipopeptides and biocontrol potential in maize. J Appl Microbiol 2020; 128: 242-254.
14. Hussain T, Khan AA. Determining the antifungal activity and characterization of Bacillus siamensis AMU03 against Macrophomina phaseolina (Tassi) Goid. Indian Phytopathol 2020; 73: 507-516.
15. Sharma A, Kaushik N, Sharma A, Bajaj A, Rasane M, Shouche YS, et al. Screening of tomato seed bacterial endophytes for antifungal activity reveals lipopeptide producing Bacillus siamensis strain NKIT9 as a potential bio-control agent. Front Microbiol 2021; 12: 609482.
16. Xie Z, Li M, Wang D, Wang F, Shen H, Sun G, et al. Biocontrol efficacy of Bacillus siamensis LZ88 against brown spot disease of tobacco caused by Alternaria alternata. Biol Control 2021; 154: 104508.
17. Pan H, Tian X, Shao M, Xie Y, Huang H, Hu J, et al. Genome mining and metabolic profiling illuminate the chemistry driving diverse biological activities of Bacillus siamensis SCSIO 05746. Appl Microbiol Biotechnol 2019; 103: 4153-4165.
18. Caulier S, Nannan C, Gillis A, Licciardi F, Bragard C, Mahillon J. Overview of the antimicrobial compounds produced by members of the Bacillus subtilis group. Front Microbiol 2019; 10: 302.
19. Kim YT, Kim SE, Lee WJ, Fumei Z, Cho MS, Moon JS, et al. Isolation and characterization of a high iturin yielding Bacillus velezensis UV mutant with improved antifungal activity. PLoS One 2020; 15(12): e0234177.
20. Zhang X, Chen X, Qiao X, Fan X, Huo X, Zhang D, et al. Isolation and yield optimization of lipopeptides from Bacillus subtilis Z-14 active against wheat take-all caused by Gaeumannomyces graminis var. tritici. J Sep Sci 2021; 44: 931-940.
21. Sun J, Liu Y, Lin F, Lu Z, Lu Y. CodY, ComA, DegU and Spo0A controlling lipopeptides biosynthesis in Bacillus amyloliquefaciens fmbJ. J Appl Microbiol 2021; 131: 1289-1304.
22. Yang R, Lei S, Xu X, Jin H, Sun H, Zhao X, et al. Key elements and regulation strategies of NRPSs for biosynthesis of lipopeptides by Bacillus. Appl Microbiol Biotechnol 2020; 104: 8077-8087.
23. Zhang Z, Ding ZT, Zhong J, ZhouJY, Shu D, Luo D, et al. Improvement of iturin A production in Bacillus subtilis ZK0 by overexpression of the comA and sigA genes. Lett Appl Microbiol 2017; 64: 452-458.24. Apimeteethamrong S,
Kittiwongwattana C. Medium effect on antagonistic activity and detection of nonribosomal peptide synthetase genes in epiphytic Bacillus strains. Curr Appl Sci Technol 2021;21: 637-651.
25. Svec D, Tichopad A, Novosadova V, Pfaffl MW, Kubista M. How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments. Biomol Detect Quantif 2015; 3: 9-16.
26. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) method. Methods 2001; 25: 402-408.
27. Reiter L, Kolstø AB, Piehler AP. Reference genes for quantitative, reverse-transcription PCR in Bacillus cereus group strains throughout the bacterial life cycle. J Microbiol Methods 2011; 86: 210-217.
28. Salzberg LI, Botella E, Hokamp K, Antelmann H, Maaß S, Becher D, et al. Genome-wide analysis of phosphorylated PhoP binding to chromosomal DNA reveals several novel features of the PhoPR-mediated phosphate limitation response in Bacillus subtilis. J Bacteriol 2015; 197: 1492-1506.
29. Rocha DJP, Santos CS, Pacheco LGC. Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis. Antonie van Leeuwenhoek 2015; 108: 685-693.
30. Higazy NS, Saleh AE, Hassan ZU, Thani RA, Migheli Q, Jaoua S. Investigation and application of Bacillus pumilus QBP344-3 in the control of Aspergillus carbonarius and ochratoxin A contamination. Food Control 2019; 119: 107464.
31. Sun D, Liao J, Sun L, Wang Y, Liu Y, Deng Q, et al. Effect of media and fermentation conditions on surfactin and iturin homologues produced by Bacillus natto NT-6: LC–MS analysis. AMB Express 2019; 9: 120.
32. Xu WF, Ren HS, Ou T, Lei T, Wei JH, Huang CS, et al. Genomic and functional characterization of the endophytic Bacillus subtilis 7PJ-16 strain, a potential biocontrol agent of mulberry fruit sclerotiniose. Microb Ecol 2019; 77: 651-663.
33. Männik J, Walker BE, Männik J. Cell cycle-dependent regulation of FtsZ in Escherichia coli in slow growth conditions. Mol Microbiol 2018; 110: 1030-1044.
34. Holtzendorff J, Partensky F, Jacquet S, Bruyant F, Marie D, Garczarek L, et al. Diel expression of cell cycle-related genes in synchronized cultures of Prochlorococcus sp. strain PCC 9511. J Bacteriol 2001; 183: 915-920.
35. Wu L, Wu H, Chen L, Xie S, Zang H, Borriss R, et al. Bacilysin from Bacillus amyloliquefaciens FZB42 has specific bactericidal activity against harmful algal bloom species. Appl Environ Microbiol 2014; 80: 7512-7520.
| Files | ||
| Issue | Vol 14 No 2 (2022) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v14i2.9188 | |
| Keywords | ||
| Bacillus; Reverse-transcription quantitative polymerase chain reaction; Gene expression; Culture media | ||
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How to Cite
1.
Nuwong W, Kittiwongwattana C. Validation of reference genes for the normalization of RT-qPCR gene expression in Bacillus siamensis 1021 grown in different culture media. Iran J Microbiol. 2022;14(2):194-202.
